Modulation of IFN action via novel regulatory factors

描述（由申请人提供）：细胞因子干扰素家族对于促进几种生理过程，如抗病毒、抗肿瘤和免疫应答至关重要。它们在临床上用于治疗许多癌症、病毒性疾病和神经退行性疾病。通过与其他细胞因子相互作用，IFN形成控制肿瘤细胞生长和宿主对病原体防御的细胞间信号分子的大网络。以前，我们已经确定了一种新的干扰素调节元件及其同源转录因子。一种这样的蛋白质是CCAAT/增强子结合蛋白-β（C/EBP-2），其是已知调节细胞分化、能量代谢、免疫应答、肿瘤生长和凋亡的转录因子。本实验室的基因表达微阵列分析确定了几个IFN调控基因，其表达需要C/EBP-2。其中之一是死亡相关蛋白激酶1（DAPK 1），它是细胞凋亡、细胞周期和转移的重要调节因子。dapk 1基因的表达在多种人类癌症中经常丢失。DAPK 1还调节自噬（一种新的死亡形式），这对于去除受损的细胞器，对抗细胞内病原体，抗原呈递和肿瘤抑制至关重要。有趣的是，小鼠中C/EBP-2基因的缺失导致了许多这些缺陷。在上一个资助期间，我们已经证明了C/EBP-2在调节DAPK 1方面的核心作用。在初步研究中，我们发现DAPK 1的表达需要其他的反式作用因子。值得注意的是，激活转录因子6（ATF 6），内质网依赖性应激反应的关键调节因子，似乎调节DAPK 1的表达。我们提出ATF 6和C/EBP-2之间的直接相互作用通过自噬导致DAPK 1表达和生长抑制，其中MAP激酶，细胞凋亡刺激激酶1（ASK 1）提供关键信号输入。在本提案的具体目标1中，我们将研究C/EBP-2和ATF 6如何协同上调DAPK 1表达。在具体目标2中，我们将研究ASK 1如何通过ATF 6控制DAPK 1表达。增强子结合的转录因子（TF）使用转录共激活因子促进转录。其中一种是由多个蛋白质组成的分子桥--介体，它将转录信号从转录因子传递到TATA盒上的一般转录因子复合体。在上一个资助期间的研究还确定了Med 1，Med 1是Mediator的主要亚基，Mediator是IFN诱导的C/EBP- 2结合伴侣。我们目前的初步证据表明，参与ZIP激酶（ZIPK），DAPK家族的成员，在调节DAPK 1的表达响应干扰素。我们假设ZIPK通过调节Med 1的C/EBP结合结构域的磷酸化来调节DAPK 1的表达。这方面将在具体目标3中进行研究。我们将使用RNAi、敲除小鼠、蛋白质相互作用、ChIP测定、诱变和转录分析来评估这些因子与DAPK 1和自噬的关键相关性。从了解这些途径中获得的知识不仅可以定义DAPK 1的关键调节因子，还可以提供DAPK 1丢失如何发生的指标。我们将研究这些途径与人类CLL的相关性，CLL是一种dapk 1似乎发挥重要肿瘤抑制作用的疾病。这些反过来将允许更好地设计治疗方法来对抗肿瘤进展和转移。 公共卫生相关性：肿瘤转移的发生是由于某些关键基因的丢失和相应生物过程的抑制。本申请中提出的研究将研究抗转移基因DAPK 1的调节，该基因对肿瘤抑制和自噬反应至关重要。

DESCRIPTION (provided by applicant): The interferon family of cytokines is critical for promoting several physiologic processes, such as antiviral, antitumor, and immune responses. They are in clinical use for the therapy of a number of cancers, viral diseases and neurodegenerative disorders. By interacting with other cytokines IFNs form a large network of intercellular signaling molecules that control neoplastic cell growth and host defenses against pathogens. Previously, we have identified a novel IFN-regulated element and its cognate transcription factors. One such protein is CCAAT/Enhancer Binding protein-beta (C/EBP-2), a transcription factor known to regulate cell differentiation, energy metabolism, immune response, tumor growth and apoptosis. A gene expression micro- array analysis in our lab identified several IFN regulated genes, whose expression required C/EBP-2. One of them is the death associated protein kinase1 (DAPK1), an important regulator of apoptosis, cell cycle, and metastasis. The expression of dapk1 gene is frequently lost in several human cancers. DAPK1 also regulates autophagy (a novel form of death), which is critical for the removal of damaged organelle, fighting intracellular pathogens, antigen presentation and tumor suppression. Interestingly, the loss of C/EBP-2 gene in mice causes many of these defects. During the last funding period, we have shown a central role for C/EBP-2 in regulating DAPK1. In preliminary studies, we show that the expression of DAPK1 requires other transacting factors. Notably, Activating transcription factor 6 (ATF6), a key regulator of endoplasmic reticulum-dependent stress responses, appears to regulate DAPK1 expression. We propose that a direct interaction between ATF6 and C/EBP-2 leads to DAPK1 expression and growth suppression via autophagy, wherein the MAP Kinase, apoptosis- stimulating kinase 1(ASK1), provides critical signal inputs. In specific aim 1 of this proposal we will investigate how C/EBP-2 and ATF6 collaborate to upregulate DAPK1 expression. In specific aim 2, we will investigate how ASK1 controls DAPK1 expression via ATF6. Enhancer bound transcription factors (TFs) promote transcription using transcriptional co-activators. One of them the Mediator, a molecular bridge comprised of multiple proteins, communicates the transcriptional signals from the TFs to general transcription factor complex at the TATA-box. Studies during the last funding period also identified Med1, a major subunit of Mediator, an IFN-induced binding partner of C/EBP- 2. We present preliminary evidence for the involvement of ZIP-kinase(ZIPK), a member of the DAPK family, in regulating DAPK1 expression in response to IFNs. We hypothesize that ZIPK regulates DAPK1 expression by modulating the phosphorylation of C/EBP-binding domain of Med1. This aspect will be investigated in specific aim 3. We will evaluate the critical relevance of these factors to DAPK1 and autophagy using RNAi, knockout mice, protein-interactions, ChIP assays, mutagenesis and transcriptional analyses. Knowledge gained from an understanding these pathways, will not only define the critical regulators of DAPK1, but also will provide indicators into how a loss of DAPK1 can occur. We will investigate the relevance of these pathways to human CLL, a disease in which dapk1 appears to play an important tumor suppressive role. These in turn will allow a better design of therapeutics to combat tumor progression and metastasis. PUBLIC HEALTH RELEVANCE: Tumor metastasis occurs due to a loss of certain critical genes and a suppression of corresponding biological processes. Studies proposed in this application will investigate the regulation of an anti-metastatic gene, DAPK1, which is critical for tumor suppression and autophagic responses.